In this study, the enzymatic biosynthesis of met5-enkephalin from endogenous precursors was investigated. A trypsin-like enzyme was partially purified from bovine adrenal chromaffin granules through the use of affinity chromatography. This enzyme preparation was able to generate met5-enkephalin from endogenous substrate(s). Met5-enkephalin production was not inhibited by sulfhydryl reagents such as p-chloromercuriphenyl sulfonate nor stimulated by dithiothreitol, suggesting that this enzyme is not a lysosomal enzyme such as cathepsin B. Enzymatic activity was strongly inhibited by several trypsin inhibitors including soybean trypsin inhibitor, aprotinin, and diisopropylfluorophosphate. These results imply that this adrenal enzyme is not a protease of lysosomal origin but is a chromaffin granular enzyme capable of generating enkephalin. The physiological role of enkephalin in adrenal is still unclear. The identification of this enkephalin generating enzyme will now enable us to study the regulation of enkephalin system in the adrenal and then, in turn, explore its physiological role. The enzyme will be further purified, characterized and a specific method will be developed to study the regulation of the adrenal enkephalin system.